1. Field of the Invention
The present invention relates to a method for synthesizing aromatic amino acids, more particularly, to a method for synthesizing homophenylalanine in a thermostable and easy process.
2. Description of the Related Art
Hypertension, also called high blood pressure, means patient's blood pressure higher than normal standard. Severely high pressure is defined as 50% or over 50% higher than standard. Persistent hypertension might lead to strokes, heart attacks, heart failure and arterial aneurysm. In industrialized countries, hypertension is a main reason of cardiovascular morbidity and mortality.
Renin-Angiotensin system (RAS) is a crucial moderating factor for blood pressure. In the RAS reaction, Angiotensin-Converting Enzyme (ACE), which is a circulating enzyme for mediating extracellular volume and arterial vasoconstriction, has two primary functions: (1) catalyzing the conversion of angiotensin I to angiotensin II as a vasoconstrictor in a substrate concentration dependent manner, and (2) catalyzing the degradation of a vasodilator-bradykinin. Therefore, ACE is a good target for medicines to therapy some multiple failures including high blood pressure, heart failure, diabetic nephropathy and type 2 diabetes mellitus. Angiotensin-Converting Enzyme Inhibitors (ACEIs) can inhibit the activity of ACE, decreasing the formation of angiotensin II and the degradation of bradykinin. Therefore, ACEIs have been used to prevent over-activation of the RAS, which leads to a systematic dilation of blood vessels and lower blood pressure. One of the raw materials for synthesizing ACEIs is homophenylalanine (HPA). HPA is sorted into a D-form homophenylalanine (D-HPA) and a L-form homophenylalanine (L-HPA). Due to its slow metabolism and tendency to remain in organisms, the safety and medicines' kinetics of the D-HPA needs further evaluation. Consequently, most medicines of ACEIs use L-HPA as a raw material, such as Enalapril, Ramipril, Quinapril and Lisinopril. HPA is not a natural occurred amino acid and can be synthesized artificially. The conventional synthesis methods of amino acids includes an enantioseletive hydrogenation method, a full chemical synthesis method and a biochemical synthesis method.
The enantioseletive hydrogenation method uses organic solvents to extract D-HPA and L-HPA from a kinetic resolution. Sequentially, D-HPA can be isolated by different optical activity, but the D-HPA is not a good raw material for current ACEIs. Therefore, the synthetic HPA by the enantiotioseletive hydrogenation method is limited.
The full chemical synthesis method is widely used in pharmaceutical industries for massive synthesis of aromatic amino acids. The full chemical synthesis method uses an intermediate product beta-benzoylacrylic acid with 1-arylethylamine to synthesize HPA through a reduction. However, the product of HPA includes D-HPA and L-HPA so that an additional separation is required to separate D-HPA and L-HPA. The full chemical synthesis method for HPA is complicated and uses many kinds of chemicals and organic solvents, usually leading to high cost and environmental pollution.
The biochemical synthesis method is a biotransformation method in which an aminotransferase is used as a biocatalyst to selectively synthesize stereospecific amino acids. The biochemical synthesis method has some advantages, such as being simple to synthesize stereospecific amino acids and using few chemicals and low environmental pollution. A previous report showed that an E. coli aspartate aminotransferases can be used to synthesize L-HPA. It uses an aspartate as an amino donor and an 2-oxo-4-phenylbutyric acid (OPBA) as an amino acceptor in the amino-transferring reaction at 37° C. However, E. coli aspartate aminotransferase has following defects in synthesizing L-HPA.
First of all, the efficiency in synthesizing amino acid by the biochemical synthesis method is low. A higher temperature leads to a higher reaction rate, but E. coli aspartate aminotransferase is less stable, and easily becomes inactive over 37° C. or after hours of working time. Hence, the E. coli aspartate aminotransferase reaction cannot be carried out at a higher temperature. Moreover, even if the E. coli aspartate aminotransferase reaction is carried out at 37° C., the E. coli aspartate aminotransferase easily becomes inactive after few hours. Therefore, a large amount of E. coli aspartate aminotransferase or a time-consuming process is required to synthesize amino acids by the biochemical synthesis method. On the other hand, a lower reaction temperature also causes a lower solubility of aromatic amino acceptors, and accordingly a lower efficiency in amino acids synthesizing reaction is obtained. Next, the cost of the biochemical synthesis method is high. E. coli aspartate aminotransferase easily becomes inactive over 37° C., so that coolers or freezers are required for its preparation, storage and also for its transportation. The enzyme activity of E. coli aspartate aminotransferase will be decreased if proper procedures are not taken in the preparation, storage and transportation. Thus, a high cost is required to obtain active E. coli aspartate aminotransferase.